摘要
Bacillus circulans chitinase A1 (ChiA1) has a deep substrate-binding cleft on top of its (β/α)8-barrel catalytic domain and an interaction between the aromatic residues in this cleft and bound oligosaccharide has been suggested. To study the roles of these aromatic residues, especially in crystalline-chitin hydrolysis, site-directed mutagenesis of these residues was carried out. Y56A and W53A mutations at subsites - 5 and - 3, respectively, selectively decreased the hydrolysing activity against highly crystalline β-chitin. W164A and W285A mutations at subsites + 1 and + 2, respectively, decreased the hydrolysing activity against crystalline β-chitin and colloidal chitin, but enhanced the activities against soluble substrates. These mutations increased the Km-value when reduced (GlcNAc)5 (where GlcNAc is N-acetylglucosamine) was used as the substrate, but decreased substrate inhibition observed with wild-type ChiA1 at higher concentrations of this substrate. In contrast with the selective effect of the other mutations, mutations of W433 and Y279 at subsite - 1 decreased the hydrolysing activity drastically against all substrates and reduced the Kcat-value, measured with 4-methylumbelliferyl chitotrioside to 0.022% and 0.59% respectively. From these observations, it was concluded that residues Y56 and W53 are only essential for crystalline-chitin hydrolysis. W164 and W285 are very important for crystalline-chitin hydrolysis and also participate in hydrolysis of other substrates. W433 and Y279 are both essential for catalytic reaction as predicted from the structure.
原文 | English |
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頁(從 - 到) | 237-244 |
頁數 | 8 |
期刊 | Biochemical Journal |
卷 | 376 |
發行號 | 1 |
DOIs | |
出版狀態 | Published - 2003 11月 15 |
All Science Journal Classification (ASJC) codes
- 生物化學
- 分子生物學
- 細胞生物學